CN114621979B - Cell mechanical transfection method and device based on flexible variable-section micro-channel - Google Patents
Cell mechanical transfection method and device based on flexible variable-section micro-channel Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
- B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
Abstract
The present disclosure provides a method of cell mechanical transfection based on flexible variable cross-section microchannels, comprising: s1, introducing liquid containing cells and exogenous substances into a liquid path layer of a microfluidic device, wherein the liquid path layer comprises a plurality of liquid micro-channels; s2, applying air pressure to an air channel layer of the microfluidic device, deforming a film layer between the liquid channel layer and the air channel layer, and changing the section size of a liquid channel microchannel at a corresponding position; when the cells pass through the liquid micro-channels at the corresponding positions, the stress is applied by the flexible film layer, the micro-wound film holes are instantaneously generated on the cell membranes, and exogenous substances enter the cells through the micro-wound film holes, so that the cell transfection is completed. The disclosure also provides a microfluidic device for cell mechanical transfection. The method and the device provided by the disclosure can avoid damage to cells caused by rigid extrusion, have high tolerance to cell size, can adapt to cell groups with large and small heterogeneity, and provide a powerful and practical mechanical transfection method for applications such as biological manufacturing, cell therapy, regenerative medicine and the like.
Description
Technical Field
The disclosure relates to the technical field of microfluidic chips, in particular to a cell mechanics transfection method and device based on a flexible variable-section microchannel.
Background
Intracellular delivery is the transport of membrane-impermeable molecules (e.g., DNA, RNA, proteins, drugs, or nanomaterials) across the cell membrane into the cytoplasm or nucleus. And delivery of substances to cells is an important step in understanding cell function and reprogramming cell behavior. Viral vectors are currently the most favored method of intracellular delivery. Because viruses utilize their own infectious pathways, they have excellent gene transfer efficiency and can be permanently transferred. However, the development is limited by the possibility of inflammatory immune response, genetic toxicity, and lower packaging capacity. As a non-viral approach, cationic lipid carriers are used to transport cargo into cells by endocytosis, but are more difficult to transfect for suspension cells etc., have a high degree of cell type dependence and some are less efficient in substance delivery. While membrane disruption techniques are based on the application of external electrical, thermal, optical or mechanical energy to cells to physically open the cell membrane, delivering external cargo dispersed in solution into the cells. Electroporation delivery is currently the most common, however electroporation cells are less viable and are prone to losing cell phenotype and function.
Microfluidic-based delivery techniques, which are not limited by cell type and nature of the delivered substance, have attracted considerable attention as an emerging solution, and microfluidic physical perforation techniques have made delivery of substances that have previously been difficult. These techniques, in turn, use shear or contractile forces to rapidly deform the cells, thereby creating transient pores in the cell membrane. However, it is not clear how the potential mechanical properties affect the efficiency of substance delivery, especially the impact of rigid extrusion of narrow tubing on cell damage. Meanwhile, the current delivery efficiency based on the microfluidic physical perforation technology is different according to cell types, different extrusion parameters need to be studied to adapt to cells of different sizes, and the method is difficult to be applied to cells with size heterogeneity. And the silicon etching channel of the current micro-fluidic device which is narrowly extruded is easy to be blocked, and the further application of the micro-fluidic device is influenced by the generated cell fragments and operation problems.
Disclosure of Invention
First, the technical problem to be solved
In view of the above problems, the present disclosure provides a cell mechanical transfection method and device based on flexible variable cross-section micro-channels, which are used for at least partially solving the technical problems that the conventional cell transfection method has large damage to cells, is difficult to adapt to cells with size heterogeneity, and micro-channels are easy to block.
(II) technical scheme
In one aspect, the disclosure provides a cell mechanical transfection method based on a flexible variable cross-section microchannel, comprising: s1, introducing liquid containing cells and exogenous substances into a liquid path layer of a microfluidic device, wherein the liquid path layer comprises a plurality of liquid micro-channels; s2, applying air pressure to an air channel layer of the microfluidic device, deforming a film layer between the liquid channel layer and the air channel layer, and changing the section size of a liquid channel microchannel at a corresponding position; when the cells pass through the liquid micro-channels at the corresponding positions, the stress is applied by the flexible film layer, the micro-wound film holes are instantaneously generated on the cell membranes, and exogenous substances enter the cells through the micro-wound film holes, so that the cell transfection is completed.
Further, S1 introducing a liquid containing cells and foreign substances into a liquid path layer of the microfluidic device includes: the cells are driven to flow and the flow rate of the liquid is regulated using a syringe pump, pressure pump, pipette, syringe or injector.
Further, applying air pressure to the air path layer of the microfluidic device in S2 includes: and applying air pressure to the air channel layer by using an air pressure pump, wherein the air pressure comprises the step of applying positive pressure to squeeze the liquid micro-channel at the corresponding position or the step of restoring the liquid micro-channel when negative pressure is loaded.
Further, S1 further includes: the microchannels are rinsed and incubated with a delivery buffer, wherein the delivery buffer comprises phosphate buffered solution, pluronic and bovine serum albumin.
Further, S1 further includes: cells were cultured and resuspended using delivery buffer.
Further, the material of the microfluidic device comprises a thermoplastic or cold-molded elastomeric material.
Further, the cells include any one or a combination of human cells, animal cells, plant cells, bacterial cells; or the cells comprise any one of suspension cells, adherent cells or a combination thereof; or the cells comprise any one of primary cells, cell lines, or a combination thereof.
Further, the foreign substance includes any one of a plasmid, DNA, RNA, amino acids, peptides, proteins, drugs, growth factors, nanomaterials, viruses, or a combination thereof.
Another aspect of the present disclosure provides a microfluidic device for implementing the aforementioned flexible variable cross-section microchannel-based cytomechanical transfection method, comprising: the liquid path layer comprises a plurality of liquid micro-channels for introducing liquid of cells and exogenous substances; the gas path layer comprises a plurality of gas micro-channels, and at least part of the gas micro-channels are aligned with the liquid micro-channels; the thin film layer is arranged between the liquid path layer and the gas path layer, when air pressure is applied to the gas path layer, the thin film layer is used for deforming and changing the section size of the liquid path micro-channel at the corresponding position, cells in the thin film layer are flexibly extruded by the thin film layer, micro-wound film holes are instantaneously formed in cell membranes, and exogenous substances enter the cells through the micro-wound film holes to finish cell transfection.
Further, the liquid micro-channels and the gas micro-channels are arranged in a staggered manner; the extrusion size of the film layer is in the range of 0-25 mu m, and the elastic modulus is in the range of 1 KPa-1 MPa.
(III) beneficial effects
According to the cell mechanical transfection method and device based on the flexible variable-section micro-channel, the air path layer is pressurized, the thin film layer deforms and flexibly extrudes the liquid micro-channel, and the cell is stressed by the flexible air film when passing through the variable-section micro-channel, so that a minimally invasive film hole is instantaneously formed in a cell membrane to allow a foreign substance to be delivered to the cell; the flexible variable-section micro-channel can flexibly and adjustably realize cell delivery with different sizes in one chip, meanwhile, the design has high tolerance to cell size and is not easy to block, and the device is simple to operate and can realize the delivery of high-flux cells.
Drawings
FIG. 1 schematically illustrates an extrusion flow diagram of a flexible variable cross-section microchannel-based cell mechanical transfection method in accordance with an embodiment of the disclosure;
FIG. 2 schematically illustrates a schematic diagram of a high-speed microscope image display cell extrusion process in accordance with an embodiment of the present disclosure;
FIG. 3 schematically shows a graph of fluorescence and cellular activity delivered after endocytosis of a 3kDa dextran control and extrusion of a microvalve in accordance with an embodiment of the present disclosure;
FIG. 4 schematically shows a schematic representation of the delivery efficiency and cell activity of modulating gas pressure deformation to optimize the delivery of 70k Da FITC-dextran by MCF-7 cells in accordance with an embodiment of the present disclosure;
FIG. 5 schematically shows a schematic of the results of delivery of 70k Da FITC-dextran into primary hard-to-transfect MEF cells in an embodiment in accordance with the present disclosure;
FIG. 6 schematically shows a schematic of the results of 2000 kDa FITC-dextran delivery into primary hard-to-transfect immune T cells in an embodiment in accordance with the present disclosure;
FIG. 7 schematically illustrates a schematic of the results of EGFP-mRNA delivery to MCF-7 cells by flexible extrusion in accordance with an embodiment of the present disclosure;
FIG. 8 schematically shows a schematic of the results of delivery of plasmid LifeAct-TagRFP to MCF-7 cells by flexible extrusion in accordance with an embodiment of the present disclosure.
Detailed Description
For the purposes of promoting an understanding of the principles and advantages of the disclosure, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same.
The present disclosure is directed to a method and microfluidic device for introducing membrane-impermeable molecules (e.g., DNA, RNA, proteins, drugs, or nanomaterials) into the cytoplasm or nucleus across the cell membrane. The method and the device can overcome the limitations of the current intracellular delivery, such as high dependence of biochemical methods such as liposome or virus on the cells and substances to be delivered. It is not clear how the potential mechanical properties affect the efficiency of substance delivery, especially the impact of rigid extrusion of narrow tubing on cell damage. Meanwhile, the current delivery efficiency based on the microfluidic physical perforation technology is different according to cell types, different extrusion parameters need to be studied to adapt to cells with different sizes, and the method is difficult to be applied to cell groups with size heterogeneity. And the silicon etching channel of the current micro-fluidic device which is narrowly extruded is easy to be blocked, and the generated cell fragments and operation problems influence a series of technical problems such as further application and the like.
To achieve the above object, the present disclosure provides a cell mechanical transfection method based on flexible variable cross-section micro-channels, please refer to fig. 1, comprising: s1, introducing liquid containing cells and exogenous substances into a liquid path layer of a microfluidic device, wherein the liquid path layer comprises a plurality of liquid micro-channels; s2, applying air pressure to an air channel layer of the microfluidic device, deforming a film layer between the liquid channel layer and the air channel layer, and changing the section size of a liquid channel microchannel at a corresponding position; when the cells pass through the liquid micro-channels at the corresponding positions, the stress is applied by the flexible film layer, the micro-wound film holes are instantaneously generated on the cell membranes, and exogenous substances enter the cells through the micro-wound film holes, so that the cell transfection is completed.
The cells and the exogenous substances flow along with the liquid in the micro-channels of the liquid path layer, when the cells pass through the micro-channels where the thin film layer is deformed, the cross-section size of the micro-channels is reduced, and the cells are stressed by the flexible air film layer, so that the micro-wound film holes are instantaneously formed on the cell film to allow the exogenous substances to be delivered into the cells or the nuclei of the cells. The cell transfection method by flexible extrusion avoids potential damage to cells by the existing rigid pipeline extrusion, and the cells after delivery still have higher activity and can keep higher delivery efficiency.
On the basis of the above embodiment, S1 introducing a liquid containing cells and foreign substances into a liquid path layer of a microfluidic device includes: the cells are driven to flow and the flow rate of the liquid is regulated using a syringe pump, pressure pump, pipette, syringe or injector.
Various methods may be utilized in the present disclosure to drive the flow of liquid-path cells, including but not limited to the five devices described above, such as automated or semi-automated liquid handling systems, and the like, which drive the flow of liquid-path cells in a gas-tight and squeeze-out delivery of the mixed liquid from the liquid-path inlet of the liquid-path layer. The flow rate of the liquid is controlled, for example, in increments of 50. Mu.l/min, and the cell flow rate is related to the transfection efficiency and the cell expression intensity.
On the basis of the above embodiment, applying air pressure to the air path layer of the microfluidic device in S2 includes: and applying air pressure to the air channel layer by using an air pressure pump, wherein the air pressure comprises the step of applying positive pressure to squeeze the liquid micro-channel at the corresponding position or the step of restoring the liquid micro-channel when negative pressure is loaded.
The pneumatic pump is connected to the air passage layer through an airtight pipe, and the pneumatic pump adjusts the extrusion deformation of the middle film layer by taking 200mbar as an increment, so as to allow the flexible extrusion of the cells flowing at high speed in the lower liquid passage. The air pressure adjustment may also include a restoration of the liquid microchannel upon release of the air path positive pressure, and a negative pressure may be applied to expand the liquid microchannel through the air path. Because the liquid micro-channel section of the device is flexible, cell mechanical transfection with near zero blockage can be realized. Besides the stress applied to the cells by the middle flexible film layer through adjusting the air pressure of the air passage layer, the middle flexible film layer can be deformed by other external forces, including but not limited to air pressure deformation, magnetic deformation, weight deformation, temperature response deformation and light response deformation.
On the basis of the above embodiment, S1 further includes: the microchannels are rinsed and incubated with a delivery buffer, wherein the delivery buffer comprises phosphate buffered solution, pluronic and bovine serum albumin.
The delivery buffer solution added with bovine serum albumin is used for slowly rinsing the micro-channel on one hand and sufficiently removing bubbles in the micro-channel; on the other hand, by incubating the microchannel, friction between the cells and the microchannel wall can be reduced while preventing cell adhesion.
On the basis of the above embodiment, S1 further includes: cells were cultured and resuspended using delivery buffer.
The delivery buffer solution is also used for re-suspending cells, can effectively reduce the adhesion between cells, reduces the risk of channel blockage, and is suitable for delivering cells and substances of most types.
On the basis of the above embodiments, the material of the microfluidic device comprises a thermoplastic or cold-molded elastomeric material.
Specifically, the liquid path layer, the gas path layer and the film layer can be prepared from PDMS materials, and of course, the materials of the liquid path layer, the gas path layer and the film layer can be the same or different.
On the basis of the above embodiments, the cells include any one of human cells, animal cells, plant cells, bacterial cells, or a combination thereof; or the cells comprise any one of suspension cells, adherent cells or a combination thereof; or the cells comprise any one of primary cells, cell lines, or a combination thereof.
The flexible squeeze delivery of the present disclosure is independent of the type of cells, including, and not limited to, the cells described above, and is not limited by cell type.
Based on the above embodiments, the foreign substance includes any one of plasmid, DNA, RNA, amino acid, peptide, protein, drug, growth factor, nanomaterial, virus, or a combination thereof.
The flexible squeeze delivery of the present disclosure is also independent of the type of exogenous material, including and not limited to the cells described above, and is not limited by the type and molecular weight of the exogenous material.
Compared with the prior art, the method has the advantages that stress is applied through the flexible film layer, potential damage to cells caused by extrusion of the existing rigid pipeline can be avoided, and the cells can maintain ultrahigh activity while delivering. The technology can flexibly and adjustably realize cell delivery with different sizes in one chip through the design of the variable-section micro-channel, and has high tolerance to cell size.
The present disclosure also provides a microfluidic device for cell transfection based on flexible extrusion, please refer to fig. 1, comprising: the liquid path layer comprises a plurality of liquid micro-channels for introducing liquid of cells and exogenous substances; the gas path layer comprises a plurality of gas micro-channels, and at least part of the gas micro-channels are aligned with the liquid micro-channels; the thin film layer is arranged between the liquid path layer and the air path layer, when air pressure is applied to the air path layer, the thin film layer is used for deforming and extruding the liquid micro-channel at the corresponding position, cells in the liquid micro-channel are flexibly extruded by the thin film layer, micro-wound film holes are instantaneously formed in cell membranes, and exogenous substances enter the cells through the micro-wound film holes to finish cell transfection.
The present disclosure relates to a microfluidic device for introducing membrane-impermeable molecule transport across a cell membrane into the cytoplasm or nucleus, the device being detachable, comprising a three-layer microfluidic chip for extrusion delivery: the liquid path layer, the film layer and the air path layer are arranged on the lower liquid path layer, a plurality of parallel liquid path channels are arranged on the lower liquid path layer, different air pressures are applied through the upper air path layer, and the deformation of the middle film layer is adjusted, so that the section size of the liquid path micro-channel at the corresponding position is changed. The present disclosure thus enables flexible extrusion with dynamically adjustable extrusion dimensions, enabling cells of different sizes to safely create transient pores in the cell membrane while maintaining high activity, allowing the delivery of foreign substances into the cell. Of course, the disclosure may also be that the air path layer is disposed on the lower layer, the liquid path layer is disposed on the upper layer, and the film layer is disposed between the liquid path layer and the air path layer, so long as the device structure capable of realizing dynamic adjustability of the liquid micro-channel by deforming and extruding the liquid micro-channel in the liquid path layer through the film layer is within the inventive concept of the disclosure.
On the basis of the embodiment, the liquid micro-channels and the gas micro-channels are mutually staggered.
Specifically, the liquid micro-channel and the gas micro-channel are mutually staggered, so that the partition extrusion can be better realized, for example, four areas of ABCD are designed and processed, the extrusion of cells is realized by staggering 20 palindromic structures and liquid path pipelines in each area, and the requirements of the optimal extrusion times of different cells are met by different partition combination regulation and control. The liquid micro-channels and the gas micro-channels can be arranged vertically in a staggered mode, and can be staggered at any angle between 0 and 90 degrees.
In addition, the variable cross-section micro-channel disclosed by the disclosure can realize cell delivery near zero blockage, is flexible and adjustable, can realize cell delivery of different sizes in one chip, and does not need to design and process chips of different sizes. The device can also realize the delivery of high-flux cells, and takes the design of a processed parallel liquid micro-channel as an example, 10 can be processed in 1 minute 6 ~10 7 Individual cells, and can further amplify the throughput by increasing the parallel channels.
On the basis of the embodiment, the liquid path channel with the height of 25 mu m is designed and processed, the extrusion size ranges from 0 mu m to 25 mu m, and the liquid path channel is suitable for most cell types.
The micro-channel section of the liquid path in the micro-fluidic device is variable, so that the cell delivery with different sizes can be realized in one chip, and the extrusion size is flexible and adjustable and comprises and is not limited to 0-25 mu m. Materials of the microfluidic device include but are not limited to PDMS or other thermoplastic and cold-molded elastic materials, and the materials are cheap and easy to process, and do not need complex operation; wherein the thickness of the film layer is 15 μm and the elastic modulus is 1 KPa-1 MPa. The microfluidic device has high tolerance to cells with size heterogeneity, and the damage to the cell activity caused by rigid extrusion is avoided by a flexible extrusion mode.
The microfluidic device has the characteristics of variable micro-channel section, high tolerance to cell size, adaptability to cell groups with large and small heterogeneity and high efficiency in a minimally invasive manner, and is particularly suitable for flexible mechanical transfection of cells.
The present disclosure is further illustrated by the following detailed description. The following examples illustrate the method and apparatus for cell mechanical transfection based on flexible variable cross-section microchannels. However, the following examples are merely illustrative of the present disclosure, and the scope of the present disclosure is not limited thereto.
1. Device for intracellular delivery and method of making same
The disclosure provides a detachable device as shown in fig. 1, which comprises a three-layer microfluidic chip for extrusion delivery, wherein a lower liquid path layer is provided with a plurality of parallel liquid path channels, and different air pressures are applied to adjust the deformation of an intermediate film layer through an upper air path layer, so that the height dynamic adjustment of the liquid micro channel at the extrusion position of the lower layer is realized, and the device is used for extrusion delivery of flexible cells.
The chip design process with the micro valve utilizes CAD drawing software to design the structures of the liquid micro channel and the gas micro channel, prints corresponding film masks, and then etches a male die of the liquid micro channel and the gas micro channel on the silicon chip through a photoetching experiment. Chips were then fabricated by reverse molding with polydimethylsiloxane (PDMS, sylgard 184). Firstly, mixing PDMS and a curing agent in a ratio of 10:1, uniformly stirring, and fully removing bubbles by using a vacuum pump under negative pressure. And pouring the PDMS mixed solution onto a positive film with a micro-channel, sufficiently removing bubbles by using a vacuum pump under negative pressure, and then putting the positive film into an oven for heating and curing for 2 hours at 75 ℃. After solidification, the PDMS solidified layers of the liquid channel and the gas channel are cut by a nicking knife and torn off, and a 19 # puncher is used for punching holes at the inlet and the outlet of the micro-channel and then placed in a clean dish for standby. The middle film layer is stirred uniformly by PDMS mixed solution with the speed of 25:1, then air bubbles are pumped by a vacuum pump, and the air bubbles are poured onto a clean silicon wafer, a spin coating machine is used for spin coating on the surface of the silicon wafer at the rotating speed of 4500rpm (depending on the thickness of the PDMS film layer), and the silicon wafer is put into an oven at the temperature of 75 ℃ for heating and curing for 2 hours after being slightly stood. And (3) placing the silicon wafer with the PDMS film layer and the PDMS cured layer of the air channel in a plasma cleaning machine to clean for 40 seconds at high frequency, aligning and bonding the film layer and the air channel layer, and placing the silicon wafer and the air channel in an oven to heat for 2 hours at 75 ℃. And then removing the gas path layer with the film layer, placing the liquid path layer in a plasma cleaner in the same way, cleaning for 40 seconds at high frequency, and placing an oven at 75 ℃ for heating for 2 hours after aligning and bonding.
2. Method for illustrating cell delivery using the device prepared in step 1
2.1 micro valve chip disinfection
To avoid the effect of RNase contamination on RNA delivery, RNase-free needs to be achieved for the delivery chip and catheter. First, diethyl dicarbonate (DEPC, biorgin) was added to deionized water in a fume hood to prepare DEPC water at a final concentration of 0.1%, and the mixture was shaken overnight at room temperature in a shaker. Then the chip to be killed and the guide pipe are placed in an aluminum lunch box, and the prepared 0.1% DEPC water is added for immersing for 2 hours at room temperature. The chip treated by DEPC water is put into an autoclave for sterilization for 30 minutes, and then is put into an oven at 80 ℃ for 2 hours for drying for standby.
2.2 surface activation of the inner conduit of the microvalve chip
The channels need to be surface activated to avoid cell adhesion. First, a delivery buffer was prepared, 1% Pluronic (Sigma) and 1% bovine serum albumin (BSA, maclin) were added to a phosphate buffer solution (PBS, invitrogen), and the mixture was placed in a shaker to be mixed uniformly, and after complete dissolution, the mixture was filtered with a 0.22 μm syringe filter and stored in a four-degree refrigerator. The syringe pump slowly rinsed the channel at a rate of 5 μl/min and incubated for 0.5h at room temperature after sufficient removal of channel air bubbles to prevent cell adhesion.
2.3 cell culture
Adherent cells are exemplified by the human breast cancer cell line MCF-7. The cells were removed from liquid nitrogen, thawed in a water bath at 37℃rapidly, and the supernatant was discarded by centrifugation at 1000rpm for 5min, with the addition of DMEM complete medium (complete medium composition including DMEM high sugar medium plus 10% FBS fetal bovine serum and 1% penicillin/streptomycin diab). Re-suspending the cells with DMEM complete medium, transferring to a 6cm dish, and placing in 5% CO at 37deg.C 2 Culturing in an incubator for 2-3 days, and performing subculture. After the cells are adhered to about 80-90%, PBS is used for washing, pancreatin is used for digestion for 1-2 minutes, 5mL of complete culture medium is added for stopping digestion, and the cells are fully blown into cell suspension. The cells were counted separately using a hemocytometer plate and 1 x 10 were aspirated 6 ~1*10 7 The individual cells were centrifuged and the supernatant was resuspended in 1mL of delivery buffer and placed on ice for later use.
Suspension cells are exemplified by human immune cell T cells. Adding 20 mu L/mL of collected human peripheral blood into Rosetteep TM Human cd8+ T Cell Enrichment Cocktail antibody, after mixing well, left to stand at room temperature for 20 minutes. PBS containing 2% FBS was gently mixed with blood at 1:1, the mixture was slowly added to the top of the density gradient Ficoll, centrifuged at 1200g for 20min at room temperature, and the middle white membrane layer was pipetted into a new centrifuge tube. The centrifuge tube was topped up with PBS containing 1% FBS, and after centrifugation at 600g for 10 minutes, if erythrocytes were present, the erythrocytes were lysed and washed once. Adding 100U/mL of IL-2-containing X-VIVO15 complete medium, adding 20ng/mL of IL-7, each 1X 10 6 The cells were frozen. When immediate use is required, 25 μl/min of activated magnetic beads (Dynabeads human T-activator CD3/CD28 beads) are taken and rinsed once with medium, and the T cells are then prepared to 1X 10 using 20ng/mL of complete medium of X-VIVO15 containing IL-2 6 cell/mL concentration, 1mL of the cell mixture per well was added to a 24-well plate for culture. Also at the time of squeeze delivery according to 1 x 10 6 ~1*10 7 After centrifugation, the suspension was resuspended in 1mL of delivery buffer and placed on ice for use.
2.4 Flexible extrusion delivery of cells
A delivery substance, such as FITC-labeled fluorescein isothiocyanate-dextran (3 k Da FITC-dextran, sigma-Aldrich) was included at a concentration of 0.3mg/ml1 x 10 formulated in advance 6 ~1*10 7 The cell delivery buffer/mL was then injected into a No. 21 blunt end syringe and connected to the chip fluid path inlet via an airtight tube having an inner diameter of 0.51mm, and the syringe pump (Harvard Apparatus, PHD 4400) adjusted the cell flow rate in increments of 50. Mu.l/min, with each fluid path channel having a size width of 25 μm and a height of 20. Mu.m. The air passage micro valve is connected to the air pressure pump through an airtight pipe with the inner diameter of 0.51mm, and the air pressure pump is used for pumping airOB1, mk 3) was adjusted in 200mbar as an increment to allow flexible extrusion of high-speed flowing cells in the lower liquid-circuit layer as shown in fig. 2, and then after incubating the recovered cells for 20 minutes at room temperature (here, incubation was to reduce turbulence to the cells, give the cells more opportunity to contact with the delivered substance and sufficient cell recovery time), PBS wash centrifugation was resuspended in cell culture medium and incubated in incubator for 24 hours before activity and transfection efficiency was assessed for cells delivered by flexible extrusion. LIVE/DEAD for cellular activity TM The cell viability/cytotoxicity kit (Invitrogen, L3224) was incubated at 37℃for 20min in a 1:1000 ratio in culture medium, centrifuged with PBS, placed on ice, imaged under confocal microscopy and staining was observed. The dextran as a foreign substance is efficiently and safely delivered into cells, as shown in fig. 3, and the cells have high activity after delivery due to less damage to the cells caused by the extrusion of the air valve while maintaining high delivery efficiency.
3. The delivery characteristics depend on various parameters of the micro-valve chip
There are many parameters that may affect substance delivery including, but not limited to, squeeze size, recovery size, surface modification at squeeze, flow rate in channel, cell concentration, substance concentration delivered, etc. The flexible extrusion chip of the micro valve disclosed by the invention has the advantages that the flexible adjustment of the air valve is realized, the chip with different extrusion sizes is not required to be designed and processed, and the extrusion parameters can be optimized on the same chip. The present disclosure deforms the film layer at the extrusion site by precisely applying pressure of different magnitudes and different waveforms at the gas path through a pump such as a gas cylinder or a compressor, thereby flexibly changing the extrusion size at one chip to optimize the delivery result, as shown in fig. 4. The MCF-7 cell 1-2atm air pressure is regulated, the transfection efficiency and the expression intensity delivered into the cell can be further improved by the reduction of the extrusion size along with the increase of the air pressure in a certain range, the flexible extrusion of the micro-valve chip can well keep the activity of the cell, and the micro-valve chip has little difference relative to the control endocytic group, so that the micro-valve chip has great application prospect for future further clinical application.
Various methods can be used in the present disclosure to drive cell flow in the fluid pathway layer, including but not limited to syringe pumps, pressure pumps, pipettes, syringes, etc., in which the cell flow rate is adjusted in 50 μl/min increments, with increasing cell flow rate, transfection efficiency and cell expression intensity increase, and delivery of the flexible extrusion relative to other extrusions has little difference in activity relative to the control endocytic group.
4. Flexible extrusion delivery is independent of cells and delivery substances
The present disclosure safely creates transient pores in the cell membrane by highly dynamically adjustable flexible squeeze cells while maintaining high cell activity, allowing passive expansion or convective delivery of foreign substances into the cell, thus the flexible squeeze delivery of the present disclosure is independent of the cell and the substance delivered. The substances delivered in the present disclosure include, but are not limited to, single substances such as DNA, RNA, proteins, drugs, or nanomaterials, and may be a mixture of substances. In this embodiment, some are FITC-labeled dextran of different sizes 3kDa, 70 kDa, 2000 kDa to mimic nucleic acids, proteins, nanoparticles, etc. In this example, the delivery efficiency of different FITC-labeled dextran of MCF-7 cells can be over 90%, and the dextran has the characteristic of flexible extrusion, and the activity of cells in endocytic groups is not changed obviously compared with that of cells in control endocytic groups. Exemplary nucleic acids in some cases of the present disclosure include, but are not limited to, EGFP mRNA (TriLink, L-7601-100), transfection of MCF-7 cells at a concentration of 2 μg/mL relative to the lower concentration of normal experiments, as shown in FIG. 7, the present disclosure is capable of delivering EGFP mRNA efficiently to MCF-7 cells by flexible extrusion, with higher EGFP expression after 24h, and transfectionThe dyeing efficiency reaches more than 90 percent. The DNA of the present disclosure includes, but is not limited to, the backbone plasmid LifeAct-TagRFP (Ibidi, p CMV -TagRFP), as shown in fig. 8, in this example, MCF-7 cells were delivered at a concentration of 5 μg/mL, and 1 μg/mL of the transfection-assisting agent Polybrene (omiset) was mixed into the delivery solution, which was found to have higher expression after 48 hours of delivery, and the delivery efficiency was also over 90%.
The generation of transient pores by flexible extrusion of the present disclosure is equally independent of the type of cells delivered, including and not limited to human cells, animal cells, plant cells, bacterial cells, applicable to suspension cells and adherent cells, primary cells and cell lines, and is not limited by cell type. The cell suspension may be a mixed and purified population of cells, and in some embodiments, cell lines MCF-7, heLa, IEC6, A549 of varying sizes ranging from 10 μm to 20 μm, etc. can have no significantly altered activity relative to control endocytic cells while having higher transfection efficiency, and thus the microfluidic chip of the present disclosure is more adaptable to cells of varying sizes. Meanwhile, for some primary hard-to-transfect cells, such as the cell embryo mouse fibroblast MEF commonly used in cell reprogramming, as shown in figure 5, the delivery efficiency can reach more than 50% while the high activity can be maintained, and the efficiency can be further improved through the flow rate. In this embodiment, primary mouse and human immune T cells, which are difficult to transfect, are included, and as shown in fig. 6, delivery efficiency is improved by more than 60% while maintaining high activity by adjusting the squeeze size of the air valve.
While the foregoing embodiments have been described in some detail for purposes of clarity of understanding, it will be understood that the foregoing embodiments are merely illustrative of the invention and are not intended to limit the invention, any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the present disclosure are intended to be included within the scope of the present disclosure.
Claims (7)
1. A method of cell mechanical transfection based on flexible variable cross-section microchannels, comprising:
s1, introducing liquid containing cells and exogenous substances into a liquid path layer of a microfluidic device, wherein the liquid path layer comprises a plurality of liquid micro-channels;
s2, applying air pressure to an air channel layer of the microfluidic device, wherein a thin film layer between the liquid channel layer and the air channel layer deforms, and the section size of a liquid channel micro-channel at a corresponding position is changed; when the cells pass through the liquid micro-channels at the corresponding positions, stress is applied by the flexible film layer, micro-invasive film holes are instantaneously formed in cell membranes, and exogenous substances enter the cells through the micro-invasive film holes to finish cell transfection;
wherein, the step S1 of introducing the liquid containing the cells and the exogenous substances into the liquid path layer of the microfluidic device comprises the following steps: driving the cell to flow and regulating the flow rate of the liquid using a syringe pump, a pressure pump, a pipette, a syringe, or a syringe; the S1 further includes: rinsing and incubating the microchannels with a delivery buffer; the exogenous substance is any one or combination of DNA, RNA, protein, nano material and virus;
the applying air pressure to the air path layer of the microfluidic device in S2 includes: and applying air pressure to the air channel layer by using an air pressure pump, wherein the air pressure comprises the step of applying positive pressure to squeeze the liquid micro-channel at the corresponding position or restoring the liquid micro-channel when negative pressure is loaded.
2. The flexible variable cross-section microchannel-based cytomechanical transfection method of claim 1, wherein the delivery buffer comprises phosphate buffer, pluronic, and bovine serum albumin.
3. The flexible variable cross-section microchannel-based cytomechanical transfection method of claim 2, wherein S1 further comprises:
culturing cells, and resuspending the cells using the delivery buffer.
4. The method of claim 1, wherein the material of the microfluidic device comprises a thermoplastic or cold-molded elastomeric material.
5. The method for cell mechanical transfection based on flexible variable cross-section micro-channels according to claim 1, characterized in that,
the cells comprise any one or combination of human cells, animal cells, plant cells and bacterial cells; or (b)
The cells comprise any one or a combination of suspension cells and adherent cells; or (b)
The cells include any one of primary cells, cell lines, or a combination thereof.
6. A microfluidic device for implementing a flexible variable cross-section microchannel-based cell mechanical transfection method according to any one of claims 1 to 5, comprising:
the liquid path layer comprises a plurality of liquid micro-channels for introducing liquid of cells and exogenous substances; wherein introducing the liquid of the cell and the exogenous material into the liquid pathway layer of the microfluidic device comprises: driving the cell to flow and regulating the flow rate of the liquid using a syringe pump, a pressure pump, a pipette, a syringe, or a syringe; further comprises: rinsing and incubating the microchannels with a delivery buffer; the exogenous substance is any one or combination of DNA, RNA, protein, nano material and virus;
the gas path layer comprises a plurality of gas micro-channels, and the gas micro-channels are at least partially aligned with the liquid micro-channels;
the thin film layer is arranged between the liquid path layer and the gas path layer, when air pressure is applied to the gas path layer, the thin film layer is used for deforming and changing the section size of a liquid path micro-channel at a corresponding position, the cells are flexibly extruded by the thin film layer, micro-invasive film holes are instantaneously formed in cell membranes, and exogenous substances enter the cells through the micro-invasive film holes to finish cell transfection; wherein applying air pressure to the air path layer of the microfluidic device comprises: and applying air pressure to the air channel layer by using an air pressure pump, wherein the air pressure comprises the step of applying positive pressure to squeeze the liquid micro-channel at the corresponding position or restoring the liquid micro-channel when negative pressure is loaded.
7. The microfluidic device of claim 6, wherein the liquid micro-channels are staggered with the gas micro-channels;
the extrusion size of the film layer is in the range of 0-25 mu m, and the elastic modulus is in the range of 1 KPa-1 MPa.
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